Flow cytometry is a technique which allows one to analyze or to sort particle analytes in a fluid medium (e.g. a liquid or a gas). Such particle analytes include small bodies like cells and bacteria and other particles such as fluorescent beads. In flow cytometers of the prior art, a fluid containing the small bodies is added to a sheath fluid forced by hydrodynamic into a small interaction region of laminar flow where particles transit one at a time, and a laser is aimed at the interaction region. The laser light, after having crossed the interaction region and interacted with the particle analyte, is received and analyzed, which gives information about the light-interacting properties (e.g. side scattering, forward scattering, fluorescence, etc. . . . ) of particles flowing within the focused sheath fluid in the interaction region. Several parameters of the particles may be studied simultaneously (e.g. the structure of the particle, the dimension of the particle, etc.) by marking the particles to be analyzed with dyes and measuring both the fluorescence emitted by those dyed particles and the intensity of the laser light beam transmitted and scattered in different directions after it has interacted with the fluid. All of the above techniques require the intervention of a skilled technician. In particular, in the case of the prior art flow cytometry techniques, a skilled technician must adjust and precisely align the laser beam so that the laser beam may efficiently interact with the particles flowing through the small interaction sheath region.
An alternative to classical sheath flow cytometer is capillary-based flow cell cytometers where the particles under test are free flowing into a capillary and a laser is used to transversally interrogate the properties of the particle analytes transiting in the capillary (see U.S. Pat. No. 7,410,809 to Goix et al.). In such systems, no sheath fluid is added and the particles are not forced to transit one at a time through the light-particle interaction region. Therefore, to minimize the occurrence of multiple particles passing at the same time through the interrogating beam, the sample containing the analyte is more diluted and operated at a flow rate comparatively much slower than in a standard flow cytometer. The beam delivery system is however similar to the one used in a standard cytometer in that it is based on bulk optical elements shaping the interrogation beam and bringing it to the interaction region. In some instances, the collection of scattered light from the interaction region is optimized by using capillaries having complex cross-section shapes to help collecting and focusing light of interest used for the characterization of the flowing analytes (see U.S. Pat. No. 7,564,542 to Ilkov).
Known flow cytometers usually employ lasers as the light source. Although lasers are generally effective in producing focused beams of sufficient intensity to excite the particles of interest and hence generate detectable fluorescence, the use of lasers can have some drawbacks. For example, the types of lasers employed in many known flow cytometers are very expensive, and thus increase the overall cost of the system. Also, because the lasers emit very high intensity light, stray light from the laser beam can interfere with the fluorescent and scattered light emanating from the particles of interest, thus adversely affecting their measurement by creating undesirable noise in the detection channels. Collection of fluorescent and scattered light is thus complicated by such source of noise.
Another known constraint is related to the beam quality of the laser requiring tight focusing to illuminate the small laminar flow region of interest where only one particle is present at a time. Homogeneous illumination over a small region requires light of high spatial quality generally obtained by the use of additional optical elements (e.g. collimation and focusing lens, filtering pinholes, etc. . . . ) adding to the cost and bulkiness of the beam delivery system.
Also related to the small size of the light-particle interaction region in a typical flow cytometer, is the fact that some lasers display intrinsic high-frequency intensity fluctuations referred to as Relative Intensity Noise (RIN). During the short transit time of the particle in the light beam, a short pulse of fluorescing and scattered light is generated. If the laser intensity changes significantly during this transit time, the amplitude of the generated pulse will also fluctuate in a manner that is not correlated to the properties of the particle but as a consequence of the temporal instability of the laser source and hence create a hard-to-interpret measurement artifact. Therefore, it is important to use a stable laser with a low RIN at the frequency corresponding to the reciprocal duration of the transit time, typically ranging from 1 MHz to 10 MHz. Similar considerations must be applied to the spatial uniformity of the laser beam in the plane of the transit region, because a spatially non-uniform beam (especially when the spatial non-uniformity is time-varying because of vibrations or thermal effects) will produce non-uniform excitation during the transit and hence result in non-uniform amplitude in the pulsed fluorescent or scattering signals generated by the interaction between the laser light and the transiting particle. This in turn leads to the same equivocal interpretation of the signals and of its origin described above. Once again, this imposes additional constraints on the quality of the laser spatial and temporal properties and complexity of the signal processing used to mitigate these artifacts.
Therefore, a need exists for an improved and simplified beam delivery system to mitigate the problems of measurement and overall bulkiness and cost of the apparatus.